NAD(P)- dependent responsive enzymes, electrodes and sensors, and methods for making and using the same

ABSTRACT

Embodiments of the present disclosure relate to NADP-dependent oxidoreductase compositions, and electrodes, sensors and systems that include the same. Also provided are methods for making the compositions and for detecting and/or measuring analytes with NADP-dependent oxidoreductase compositions.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority based on U.S. Provisional ApplicationNo. 62/303,626, filed Mar. 4, 2016, the disclosures of which isincorporated by reference herein in its entirety.

INTRODUCTION

The characterization of analytes in biological fluids has become anintegral part of medical diagnoses and assessments of overall health andwellness of a patient. Regularly monitoring the concentration ofparticular analytes in body fluid of a subject is becoming increasinglyimportant where the results may play a prominent role in the treatmentprotocol of a patient in a variety of disease conditions. A number ofsystems are available that analyze an analyte in a bodily fluid, such asblood, plasma, serum, interstitial fluid, urine, tears, and saliva. Suchsystems monitor the level of particular medically relevant analytes,such as, blood sugars, e.g., glucose, cholesterol, ketones, vitamins,proteins, and various metabolites. In response to this growingimportance of analyte monitoring, a variety of analyte detectionprotocols and devices for laboratory, clinical and at-home use have beendeveloped.

Nicotinamide adenine dinucleotide (NAD(P)⁺) is a coenzyme found in allliving cells. There are many biological molecules that are oxidized byNAD(P)⁺-dependent dehydrogenases. For example, glucose can be oxidizedby NAD-dependent glucose dehydrogenase, alcohol can be oxidized byNAD-dependent alcohol dehydrogenase, β-Hydroxybutyrate can be oxidizedby NAD-dependent D-3-Hydroxybutyrate dehydrogenase, etc. Development ofimproved sensors for measuring analytes that are oxidized by a varietyof different enzymes having a high degree of stability and sensitivityis desirable.

SUMMARY

Embodiments of the present disclosure relate to enzyme compositions,electrodes, sensors, methods for fabricating an analyte sensor as wellas methods for monitoring an analyte concentration in a subject with thesubject sensors. Disclosed herein are enzyme compositions that includeone or more of nicotinamide adenine dinucleotide phosphate (NAD(P)⁺) orderivative thereof, an NAD(P)⁺-dependent dehydrogenase, an NAD(P)Hoxidoreductase and an electron transfer agent having a transition metalcomplex. In certain embodiments, enzyme compositions include NAD(P)⁺ ora derivative thereof and an electron transfer agent having a transitionmetal complex. The subject enzyme compositions may also include apolymer, such as a heterocycle-containing polymer, and a crosslinker forimmobilizing the enzyme composition onto a surface (e.g., on the surfaceof an electrode). Some or all of these components may be unbound orunconnected, or two or more of these components may be bound orconnected together. For example, one or more of the nicotinamide adeninedinucleotide phosphate (NAD(P)+) or derivative thereof,NAD(P)+-dependent dehydrogenase, NAD(P)H oxidoreductase and redoxmediator are immobilized on the surface by the polymer. In certaininstances, one or more of the nicotinamide adenine dinucleotidephosphate (NAD(P)⁺) or derivative thereof, NAD(P)⁺-dependentdehydrogenase, NAD(P)H oxidoreductase and redox mediator are covalentlybonded to the polymer. In certain embodiments, the NAD(P)⁺-dependentdehydrogenase is glucose dehydrogenase, alcohol dehydrogenase orD-3-hydroxybutyrate dehydrogenase. In some embodiments, the NAD(P)Hoxidoreductase is diaphorase. Also provided are analyte sensors havingthe subject enzyme compositions disposed proximate to a workingelectrode for monitoring an analyte level in a subject as well asmethods for fabricating an analyte sensor and methods for using theanalyte sensors in monitoring an analyte, such as glucose, an alcohol orβ-hydroxybutyrate in a subject.

BRIEF DESCRIPTION OF THE DRAWINGS

A detailed description of various embodiments of the present disclosureis provided herein with reference to the accompanying drawings, whichare briefly described below. The drawings are illustrative and are notnecessarily drawn to scale. The drawings illustrate various embodimentsof the present disclosure and may illustrate one or more embodiment(s)or example(s) of the present disclosure in whole or in part. A referencenumeral, letter, and/or symbol that is used in one drawing to refer to aparticular element may be used in another drawing to refer to a likeelement.

FIG. 1 shows the signal output for a D-3-hydroxybutyrate dehydrogenasesensor over the course of 2.3 hours at varying concentrations ofD-3-hydroxybutyrate according to certain embodiments.

FIG. 2 depicts the linearity of the sensor signal of aD-3-hydroxybutyrate dehydrogenase sensor as a function ofD-3-hydroxybutyrate concentration.

FIG. 3 shows the signal output for a D-3-hydroxybutyrate dehydrogenasesensor using free NAD over the course of 3.6 hours at varyingconcentrations of D-3-hydroxybutyrate according to certain embodiments.

FIG. 4 depicts the linearity of the sensor signal of aD-3-hydroxybutyrate dehydrogenase sensor as a function ofD-3-hydroxybutyrate concentration (Ketone).

FIG. 5 depicts the stability of the sensor signal of aD-3-hydroxybutyrate dehydrogenase sensor.

FIG. 6 depicts the stability of the sensor signal of a free NAD andimmobilized NAD sensor.

DETAILED DESCRIPTION

Before the embodiments of the present disclosure are described, it is tobe understood that this invention is not limited to particularembodiments described, as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the embodiments of the invention will beembodied by the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassedwithin the invention. The upper and lower limits of these smaller rangesmay independently be included or excluded in the range, and each rangewhere either, neither or both limits are included in the smaller rangesis also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

In the description of the invention herein, it will be understood that aword appearing in the singular encompasses its plural counterpart, and aword appearing in the plural encompasses its singular counterpart,unless implicitly or explicitly understood or stated otherwise. Merelyby way of example, reference to “an” or “the” “enzymes” encompasses asingle enzyme, as well as a combination and/or mixture of two or moredifferent enzymes, reference to “a” or “the” “concentration value”encompasses a single concentration value, as well as two or moreconcentration values, and the like, unless implicitly or explicitlyunderstood or stated otherwise. Further, it will be understood that forany given component described herein, any of the possible candidates oralternatives listed for that component, may generally be usedindividually or in combination with one another, unless implicitly orexplicitly understood or stated otherwise. Additionally, it will beunderstood that any list of such candidates or alternatives is merelyillustrative, not limiting, unless implicitly or explicitly understoodor stated otherwise.

Various terms are described below to facilitate an understanding of theinvention. It will be understood that a corresponding description ofthese various terms applies to corresponding linguistic or grammaticalvariations or forms of these various terms. It will also be understoodthat the invention is not limited to the terminology used herein, or thedescriptions thereof, for the description of particular embodiments.Merely by way of example, the invention is not limited to particularlactates, bodily or tissue fluids, blood or capillary blood, or sensorconstructs or usages, unless implicitly or explicitly understood orstated otherwise, as such may vary.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the application. Nothing hereinis to be construed as an admission that the embodiments of the inventionare not entitled to antedate such publication by virtue of priorinvention. Further, the dates of publication provided may be differentfrom the actual publication dates which may need to be independentlyconfirmed.

The present disclosure discloses enzyme compositions that includenicotinamide adenine dinucleotide phosphate (NAD(P)+) or derivativethereof and an electron transfer agent having a transition metalcomplex. In some embodiments, the subject enzyme compositions includenicotinamide adenine dinucleotide phosphate (NAD(P)+) or derivativethereof, an NAD(P)+-dependent dehydrogenase, an NAD(P)H oxidoreductaseand an electron transfer agent having a transition metal complex, andanalyte sensors with enzyme layers that include immobilized NAD(P)+ orderivative thereof and an electron transfer agent comprising atransition metal complex. Embodiments of the present disclosure relateto enzyme compositions for analyte sensing including where the subjectcompositions provide for monitoring of an analyte in vivo over anextended period of time. Where the subject enzyme compositions includean NAD(P)+-dependent dehydrogenase, analyte sensors described hereinprovide for clinically accurate electrochemical measurement of analytesthat are catalyzed by an NAD(P)+-dependent dehydrogenase. As describedin greater detail below, the subject enzyme compositions provide forclinically accurate electrochemical measurement of analytes as measuredby Clark error grid analysis and/or MARD analysis and/or MAD analysis.In particular, the subject enzyme compositions provide for measurementby an analyte sensor incorporating the subject compositions to produce asignal that increases linearly as a function of analyte concentration.In addition, the subject enzyme compositions provide for clinicallyaccurate electrochemical measurement of analytes that are catalyzed byan NAD(P)+-dependent dehydrogenase within 30 seconds of contacting afluidic sample (e.g., interstitial fluid when the sensor is positionedbeneath the surface of a subject's skin) with the sensor. In certaininstances, the subject enzyme compositions provide for clinicallyaccurate electrochemical measurements of analytes that are catalyzed byan NAD(P)+-dependent dehydrogenase immediately after contacting thefluidic sample with the sensor.

The subject enzyme compositions include an NAD(P)+-dependentdehydrogenase, such as glucose dehydrogenase, alcohol dehydrogenase orD-3-hydroxybutyrate dehydrogenase. In some embodiments,NAD(P)+-dependent dehydrogenases of interest are oxidoreductasesbelonging to the enzyme class 1.1.1-

The NAD(P)+-dependent dehydrogenase may be present in the subjectcompositions in an amount that varies, such as from 0.05 μg to 5 μg,such as from 0.1 μg to 4 μg, such as from 0.2 μg to 3 μg and includingfrom 0.5 μg to 2 μg. As such, the amount of NAD(P)+-dependentdehydrogenase is from 0.01% to 10% by weight of the total enzymecomposition, such as from 0.05% to 9.5% by weight, such as from 0.1% to9% by weight, such as 0.5% to 8.5% by weight, such as from 1% to 8% byweight and including from 2% to 7% by weight of the total enzymecomposition.

Enzyme compositions also include nicotinamide adenine dinucleotidephosphate (NAD(P)+) or derivative thereof. In some embodiments, enzymecompositions of interest include nicotinamide adenine dinucleotidephosphate (NAD(P)+). In other embodiments, enzyme compositions include aderivative of nicotinamide adenine dinucleotide phosphate (NAD(P)+).Derivatives of nicotinamide adenine dinucleotide phosphate (NAD(P)+) mayinclude compounds of Formula I:

where X is alkyl, substituted alkyl, aryl, substituted aryl, acyl, andaminoacyl.

In some embodiments, X is an aminoacyl substituted alkyl. In someembodiments, X is CH₂C(O)NH(CH₂)_(y)NH₂ where y is an integer from 1 to10, such as 2 to 9, such as 3 to 8 and including where y is 6. Incertain instances, X is CH₂C(O)NH(CH₂)₆NH₂. In these embodiments, thederivative of nicotinamide adenine dinucleotide phosphate (NAD(P)⁺) inthe subject enzyme composition is:

Embodiments of the enzyme composition also include an NAD(P)Hoxidoreductase. In certain embodiments, the enzyme composition includesdiaphorase. The amount of NAD(P)H oxidoreductase (e.g., diaphorase)present in the subject compositions ranges from 0.01 μg to 10 μg, suchas from 0.02 μg to 9 μg, such as from 0.03 μg to 8 μg, such as from 0.04μg to 7 μg, such as from 0.05 μg to 5 μg, such as from 0.1 μg to 4 μg,such as from 0.2 □g to 3 □g and including from 0.5 □g to 2 □g. As such,the amount of NAD(P)H oxidoreductase (e.g., diaphorase) is from 0.01% to10% by weight of the total enzyme composition, such as from 0.05% to9.5% by weight, such as from 0.1% to 9% by weight, such as 0.5% to 8.5%by weight, such as from 1% to 8% by weight and including from 2% to 7%by weight of the total enzyme composition.

In some embodiments, the weight ratio of NAD(P)+-dependent dehydrogenaseto NAD(P)H oxidoreductase (e.g., diaphorase) ranges from 1 to 10NAD(P)+-dependent dehydrogenase to NAD(P)H oxidoreductase, such as from1 to 8, such as from 1 to 5, such as from 1 to 2 and including from 1 to1 NAD(P)+-dependent dehydrogenase to NAD(P)H oxidoreductase. In otherembodiments, the weight ratio of NAD(P)+-dependent dehydrogenase toNAD(P)H oxidoreductase ranges from 10 to 1 NAD(P)+-dependentdehydrogenase to NAD(P)H oxidoreductase, such as from 8 to 1, such asfrom 5 to 1 and including from 2 to 1 NAD(P)+-dependent dehydrogenase toNAD(P)H oxidoreductase.

Enzyme compositions of interest also include an electron transfer agenthaving an transition metal complex. They may be electroreducible andelectrooxidizable ions or molecules having redox potentials that are afew hundred millivolts above or below the redox potential of thestandard calomel electrode (SCE). Examples of transition metal complexesinclude metallocenes including ferrocene, hexacyanoferrate (III),ruthenium hexamine, etc. Additional examples include those described inU.S. Pat. Nos. 6,736,957, 7,501,053 and 7,754,093, the disclosures ofeach of which are incorporated herein by reference in their entirety.

In some embodiments, electron transfer agents are osmium transitionmetal complexes with one or more ligands, each ligand having anitrogen-containing heterocycle such as 2,2′-bipyridine,1,10-phenanthroline, 1-methyl, 2-pyridyl biimidazole, or derivativesthereof. The electron transfer agents may also have one or more ligandscovalently bound in a polymer, each ligand having at least onenitrogen-containing heterocycle, such as pyridine, imidazole, orderivatives thereof. One example of an electron transfer agent includes(a) a polymer or copolymer having pyridine or imidazole functionalgroups and (b) osmium cations complexed with two ligands, each ligandcontaining 2,2′-bipyridine, 1,10-phenanthroline, or derivatives thereof,the two ligands not necessarily being the same. Some derivatives of2,2′-bipyridine for complexation with the osmium cation include but arenot limited to 4,4′-dimethyl-2,2′-bipyridine and mono-, di-, andpolyalkoxy-2,2′-bipyridines, including 4,4′-dimethoxy-2,2′-bipyridine.Derivatives of 1,10-phenanthroline for complexation with the osmiumcation include but are not limited to 4,7-dimethyl-1,10-phenanthrolineand mono, di-, and polyalkoxy-1,10-phenanthrolines, such as4,7-dimethoxy-1,10-phenanthroline. Polymers for complexation with theosmium cation include but are not limited to polymers and copolymers ofpoly(1-vinyl imidazole) (referred to as “PVI”) and poly(4-vinylpyridine) (referred to as “PVP”). Suitable copolymer substituents ofpoly(1-vinyl imidazole) include acrylonitrile, acrylamide, andsubstituted or quaternized N-vinyl imidazole, e.g., electron transferagents with osmium complexed to a polymer or copolymer of poly(1-vinylimidazole).

The subject enzyme compositions may be heterogeneous or homogenous. Insome embodiments, each component (i.e., nicotinamide adeninedinucleotide phosphate (NAD(P)+) or derivative thereof, anNAD(P)+-dependent dehydrogenase, an NAD(P)H oxidoreductase and anelectron transfer agent having a transition metal complex) is uniformlydistributed throughout the composition, e.g., when applied to anelectrode, as described in greater detail below. For example, each ofnicotinamide adenine dinucleotide phosphate (NAD(P)+) or derivativethereof, an NAD(P)+-dependent dehydrogenase, an NAD(P)H oxidoreductaseand an electron transfer agent having a transition metal complex may bedistributed uniformly throughout the composition, such that theconcentration of each component is the same throughout.

In certain embodiments, the subject enzyme compositions described hereinare polymeric. Polymers that may be used may be branched or unbranchedand may be homopolymers formed from the polymerization of a single typeof monomer or heteropolymers that include two or more different types ofmonomers. Heteropolymers may be copolymers where the copolymer hasalternating monomer subunits, or in some cases, may be block copolymers,which include two or more homopolymer subunits linked by covalent bonds(e.g, diblock or triblock copolymers). In some embodiments, the subjectenzyme compositions include a heterocycle-containing polymer. The termheterocycle (also referred to as “heterocyclcyl”) is used herein in itsconventional sense to refer to any cyclic moiety which includes one ormore heteroatoms (i.e., atoms other than carbon) and may include, butare not limited to N, P, O, S, Si, etc. Heterocycle-containing polymersmay be heteroalkyl, heteroalkanyl, heteroalkenyl and heteroalkynyl aswell as heteroaryl or heteroarylalkyl.

“Heteroalkyl, Heteroalkanyl, Heteroalkenyl and Heteroalkynyl” bythemselves or as part of another substituent refer to alkyl, alkanyl,alkenyl and alkynyl groups, respectively, in which one or more of thecarbon atoms (and any associated hydrogen atoms) are independentlyreplaced with the same or different heteroatomic groups. Typicalheteroatomic groups which can be included in these groups include, butare not limited to, —O—, —S—, —S—S—, —O—S—, —NR37R38-, ═N—N═, —N═N—,—N═N—NR39R40, —PR41-, —P(O)2-, —POR42-, —O—P(O)2-, —S—O—, —S—(O)—,—SO2-, —SnR43R44- and the like, where R37, R38, R39, R40, R41, R42, R43and R44 are independently hydrogen, alkyl, substituted alkyl, aryl,substituted aryl, arylalkyl, substituted arylalkyl, cycloalkyl,substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl,heteroalkyl, substituted heteroalkyl, heteroaryl, substitutedheteroaryl, heteroarylalkyl or substituted heteroarylalkyl.

“Heteroaryl” by itself or as part of another substituent, refers to amonovalent heteroaromatic radical derived by the removal of one hydrogenatom from a single atom of a heteroaromatic ring system. Typicalheteroaryl groups include, but are not limited to, groups derived fromacridine, arsindole, carbazole, β-carboline, chromane, chromene,cinnoline, furan, imidazole, indazole, indole, indoline, indolizine,isobenzofuran, isochromene, isoindole, isoindoline, isoquinoline,isothiazole, isoxazole, naphthyridine, oxadiazole, oxazole, perimidine,phenanthridine, phenanthroline, phenazine, phthalazine, pteridine,purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine,pyrrole, pyrrolizine, quinazoline, quinoline, quinolizine, quinoxaline,tetrazole, thiadiazole, thiazole, thiophene, triazole, xanthene,benzodioxole and the like. In certain embodiments, the heteroaryl groupis from 5-20 membered heteroaryl. In certain embodiments, the heteroarylgroup is from 5-10 membered heteroaryl. In certain embodiments,heteroaryl groups are those derived from thiophene, pyrrole,benzothiophene, benzofuran, indole, pyridine, quinoline, imidazole,oxazole and pyrazine.

“Heteroarylalkyl” by itself or as part of another substituent, refers toan acyclic alkyl radical in which one of the hydrogen atoms bonded to acarbon atom, typically a terminal or sp3 carbon atom, is replaced with aheteroaryl group. Where specific alkyl moieties are intended, thenomenclature heteroarylalkanyl, heteroarylalkenyl and/orheterorylalkynyl is used. In certain embodiments, the heteroarylalkylgroup is a 6-30 membered heteroarylalkyl, e.g., the alkanyl, alkenyl oralkynyl moiety of the heteroarylalkyl is 1-10 membered and theheteroaryl moiety is a 5-20-membered heteroaryl. In certain embodiments,the heteroarylalkyl group is 6-20 membered heteroarylalkyl, e.g., thealkanyl, alkenyl or alkynyl moiety of the heteroarylalkyl is 1-8membered and the heteroaryl moiety is a 5-12-membered heteroaryl.

In some embodiments, the heterocycle-containing component is an aromaticring system. “Aromatic Ring System” by itself or as part of anothersubstituent, refers to an unsaturated cyclic or polycyclic ring systemhaving a conjugated π electron system. Specifically included within thedefinition of “aromatic ring system” are fused ring systems in which oneor more of the rings are aromatic and one or more of the rings aresaturated or unsaturated, such as, for example, fluorene, indane,indene, phenalene, etc. Typical aromatic ring systems include, but arenot limited to, aceanthrylene, acenaphthylene, acephenanthrylene,anthracene, azulene, benzene, chrysene, coronene, fluoranthene,fluorene, hexacene, hexaphene, hexalene, as-indacene, s-indacene,indane, indene, naphthalene, octacene, octaphene, octalene, ovalene,penta-2,4-diene, pentacene, pentalene, pentaphene, perylene, phenalene,phenanthrene, picene, pleiadene, pyrene, pyranthrene, rubicene,triphenylene, trinaphthalene and the like.

“Heteroaromatic Ring System” by itself or as part of anothersubstituent, refers to an aromatic ring system in which one or morecarbon atoms (and any associated hydrogen atoms) are independentlyreplaced with the same or different heteroatom. Typical heteroatoms toreplace the carbon atoms include, but are not limited to, N, P, O, S,Si, etc. Specifically included within the definition of “heteroaromaticring systems” are fused ring systems in which one or more of the ringsare aromatic and one or more of the rings are saturated or unsaturated,such as, for example, arsindole, benzodioxan, benzofuran, chromane,chromene, indole, indoline, xanthene, etc. Typical heteroaromatic ringsystems include, but are not limited to, arsindole, carbazole,β-carboline, chromane, chromene, cinnoline, furan, imidazole, indazole,indole, indoline, indolizine, isobenzofuran, isochromene, isoindole,isoindoline, isoquinoline, isothiazole, isoxazole, naphthyridine,oxadiazole, oxazole, perimidine, phenanthridine, phenanthroline,phenazine, phthalazine, pteridine, purine, pyran, pyrazine, pyrazole,pyridazine, pyridine, pyrimidine, pyrrole, pyrrolizine, quinazoline,quinoline, quinolizine, quinoxaline, tetrazole, thiadiazole, thiazole,thiophene, triazole, xanthene and the like.

In certain embodiments, enzyme compositions of interest include aheterocyclic nitrogen containing component, such as polymers ofpolyvinylpyridine (PVP) and polyvinylimidazole.

The polymeric enzyme compositions may also include one or morecrosslinkers (crosslinking agent) such that the polymeric backboneenzyme composition is crosslinked. As described herein, reference tolinking two or more different polymers together is intermolecularcrosslinking, whereas linking two more portions of the same polymer isintramolecular crosslinking. In embodiments of the present disclosure,crosslinkers may be capable of both intermolecular and intramolecularcrosslinkings at the same time.

Suitable crosslinkers may be bifunctional, trifunctional ortetrafunctional, each having straight chain or branched structures.Crosslinkers having branched structures include a multi-arm branchingcomponent, such as a 3-arm branching component, a 4-arm branchingcomponent, a 5-arm branching component, a 6-arm branching component or alarger number arm branching component, such as having 7 arms or more,such as 8 arms or more, such as 9 arms or more, such as 10 arms or moreand including 15 arms or more. In certain instances, the multi-armbranching component is a multi-arm epoxide, such as 3-arm epoxide or a4-arm epoxide. Where the multi-arm branching component is a multi-armepoxide, the multi-arm branching component may be a polyethylene glycol(PEG) multi-arm epoxide or a non-polyethylene glycol (non-PEG) multi-armepoxide. In some embodiments, the multi-arm branching component is anon-PEG multi-arm epoxide. In other embodiments, the multi-arm branchingcomponent is a PEG multi-arm epoxide. In certain embodiments, themulti-arm branching component is a 3-arm PEG epoxide or a 4-arm PEGepoxide.

Examples of crosslinkers include but are not limited to polyethyleneglycol diglycidyl ether, N,N-diglycidyl-4-glycidyloxyaniline as well asnitrogen-containing multi-functional crosslinkers having the structures:

In some instances, one or more bonds with the one or more components ofthe enzyme composition may be formed such as between one or more of thenicotinamide adenine dinucleotide phosphate (NAD(P)+) or derivativethereof, NAD(P)+-dependent dehydrogenase, NAD(P)H oxidoreductase andelectron transfer agent. By bonds is meant any type of an interactionbetween atoms or molecules that allows chemical compounds to formassociations with each other, such as, but not limited to, covalentbonds, ionic bonds, dipole-dipole interactions, hydrogen bonds, Londondispersion forces, and the like. For example, in situ polymerization ofthe enzyme compositions can form crosslinks between the polymers of thecomposition and the NAD(P)+-dependent dehydrogenase, nicotinamideadenine dinucleotide phosphate (NAD(P)+) or derivative thereof, theNAD(P)H oxidoreductase and the electron transfer agent. In certainembodiments, crosslinking of the polymer to the one or more of theNAD(P)+-dependent dehydrogenase, nicotinamide adenine dinucleotidephosphate (NAD(P)+) or derivative thereof, the NAD(P)H oxidoreductaseand the electron transfer agent facilitates a reduction in theoccurrence of delamination of the enzyme compositions from an electrode.

As described herein, the subject enzyme may be used in an analyte sensorto monitor the concentration of an NAD(P)+-dependent dehydrogenaseanalyte, such as glucose, an alcohol, a ketone, lactate, orβ-hydroxybutyrate, and the sensor may have one or more electrodes withthe enzyme composition. In embodiments, the analyte sensor includes: aworking electrode having a conductive material the subject enzymecomposition proximate to (e.g., disposed on) and in contact with theconductive material. One or more other electrode may be included such asone or more counter electrodes, one or more reference electrodes and/orone or more counter/reference electrodes.

The particular configuration of electrochemical sensors may depend onthe use for which the analyte sensor is intended and the conditionsunder which the analyte sensor will operate. In certain embodiments ofthe present disclosure, analyte sensors are in vivo wholly positionedanalyte sensors or transcutaneously positioned analyte sensorsconfigured for in vivo positioning in a subject. In one example, atleast a portion of the sensor may be positioned in the subcutaneoustissue for testing lactate concentrations in interstitial fluid. Inanother example, at least a portion of the sensor may be positioned inthe dermal tissue for testing analyte concentration in dermal fluid.

In embodiments, one or more of the subject enzyme compositions ispositioned proximate to (e.g., disposed on) the surface of a workingelectrode. In some instances, a plurality of enzyme compositions arepositioned proximate to the surface of working electrode (e.g., in theform of spots). In certain cases, a discontinuous or continuousperimeter is formed around each of the plurality of enzyme compositionspositioned proximate to the surface of the working electrode. Examplesof depositing a plurality of reagent compositions to the surface of anelectrode as well as forming a discontinuous or continuous perimeteraround each reagent composition is described in U.S. Patent PublicationNo. 2012/0150005 and in co-pending U.S. patent application Ser. No.62/067,813, the disclosures of which are herein incorporated byreference.

The subject enzyme compositions having nicotinamide adenine dinucleotidephosphate (NAD(P)+) or derivative thereof, NAD(P)+-dependentdehydrogenase, NAD(P)H oxidoreductase and electron transfer agent may bedeposited onto the surface of the working electrode as one largeapplication which covers the desired portion of the working electrode orin the form of an array of a plurality of enzyme compositions, e.g.,spaced apart from each other. Depending upon use, any or all of theenzyme compositions in the array may be the same or different from oneanother. For example, an array may include two or more, 5 or more enzymecomposition array features containing nicotinamide adenine dinucleotidephosphate (NAD(P)+) or derivative thereof, NAD(P)+-dependentdehydrogenase, NAD(P)H oxidoreductase and electron transfer agent, 10 ormore, 25 or more, 50 or more, 100 or more, or even 1000 or more, in anarea of 100 mm² or less, such as 75 mm² or less, or 50 mm² or less, forinstance 25 mm² or less, or 10 mm² or less, or 5 mm² or less, such as 2mm² or less, or 1 mm² or less, 0.5 mm² or less, or 0.1 mm² or less.

The shape of deposited enzyme composition may vary within or betweensensors. For example, in certain embodiments, the deposited membrane iscircular. In other embodiments, the shape will be of a triangle, square,rectangle, circle, ellipse, or other regular or irregular polygonalshape (e.g., when viewed from above) as well as other two-dimensionalshapes such as a circle, half circle or crescent shape. All or a portionof the electrode may be covered by the enzyme composition, such as 5% ormore, such as 25% or more, such as 50% or more, such as 75% or more andincluding 90% or more. In certain instances, the entire electrodesurface is covered by the enzyme composition (i.e., 100%).

Fabricating an electrode and/or sensor according to embodiments of thepresent disclosure produces a reproducible enzyme composition depositedon the surface of the electrode. For example, enzyme compositionsprovided herein may deviate from each other by 5% or less, such as by 4%or less, such as by 3% or less, such as by 2% or less, such as by 1% orless and including by 0.5% or less. In some embodiments, the sensingcomposition includes nicotinamide adenine dinucleotide phosphate(NAD(P)+) or derivative thereof and an electron transfer agent. Incertain embodiments, deposited enzyme compositions containingnicotinamide adenine dinucleotide phosphate (NAD(P)+) or derivativethereof, NAD(P)+-dependent dehydrogenase, NAD(P)H oxidoreductase andelectron transfer agent show no deviation from one another and areidentical.

In certain embodiments, methods further include drying the enzymecomposition deposited on the electrode. Drying may be performed at roomtemperature, at an elevated temperature, as desired, such as at atemperature ranging from 25° C. to 100° C., such as from 30° C. to 80°C. and including from 40° C. to 60° C.

Examples of configurations for the subject analyte sensors and methodsfor fabricating them may include, but are not limited to, thosedescribed in U.S. Pat. Nos. 6,175,752, 6,134,461, 6,579,690, 6,605,200,6,605,201, 6,654,625, 6,746,582, 6,932,894, 7,090,756, 5,356,786,6,560,471, 5,262,035, 6,881,551, 6,121,009, 6,071,391, 6,377,894,6,600,997, 6,514,460, 5,820,551, 6,736,957, 6,503,381, 6,676,816,6,514,718, 5,593,852, 6,284,478, 7,299,082, 7,811,231, 7,822,5578,106,780, and 8,435,682; U.S. Patent Application Publication Nos.2010/0198034, 2010/0324392, 2010/0326842, 2007/0095661, 2010/0213057,2011/0120865, 2011/0124994, 2011/0124993, 2010/0213057, 2011/0213225,2011/0126188, 2011/0256024, 2011/0257495, 2012/0157801, 2012/0245447,2012/0157801, 2012/0323098, and 20130116524, the disclosures of each ofwhich are incorporated herein by reference in their entirety.

In some embodiments, in vivo sensors may include an insertion tippositionable below the surface of the skin, e.g., penetrating throughthe skin and into, e.g., the subcutaneous space, in contact with theuser's biological fluid such as interstitial fluid. Contact portions ofworking electrode, a reference electrode and a counter electrode arepositioned on the first portion of the sensor situated above the skinsurface. A working electrode, a reference electrode and a counterelectrode are positioned at the inserted portion of the sensor. Tracesmay be provided from the electrodes at the tip to a contact configuredfor connection with sensor electronics.

In certain embodiments, the working electrode and counter electrode ofthe sensor as well as dielectric material of are layered. For example,the sensor may include a non-conductive material layer, and a firstconductive layer such as conductive polymer, carbon, platinum-carbon,gold, etc., disposed on at least a portion of the non-conductivematerial layer (as described above). The enzyme composition ispositioned on one or more surfaces of the working electrode, or mayotherwise be directly or indirectly contacted to the working electrode.A first insulation layer, such as a first dielectric layer may disposedor layered on at least a portion of a first conductive layer and asecond conductive layer may be positioned or stacked on top of at leasta portion of a first insulation layer (or dielectric layer). The secondconductive layer may be a reference electrode. A second insulationlayer, such as a second dielectric layer may be positioned or layered onat least a portion of the second conductive layer. Further, a thirdconductive layer may be positioned on at least a portion of the secondinsulation layer and may be a counter electrode. Finally, a thirdinsulation layer may be disposed or layered on at least a portion of thethird conductive layer. In this manner, the sensor may be layered suchthat at least a portion of each of the conductive layers is separated bya respective insulation layer (for example, a dielectric layer).

In other embodiments, some or all of the electrodes may be provided in aco-planar manner such that two or more electrodes may be positioned onthe same plane (e.g., side-by side (e.g., parallel) or angled relativeto each other) on the material. For example, co-planar electrodes mayinclude a suitable spacing there between and/or include a dielectricmaterial or insulation material disposed between the conductivelayers/electrodes. Furthermore, in certain embodiments one or more ofthe electrodes may be disposed on opposing sides of the non-conductivematerial. In such embodiments, electrical contact may be on the same ordifferent sides of the non-conductive material. For example, anelectrode may be on a first side and its respective contact may be on asecond side, e.g., a trace connecting the electrode and the contact maytraverse through the material. A via provides an avenue through which anelectrical trace is brought to an opposing side of a sensor.

The subject analyte sensors be configured for monitoring the level of ananalyte (e.g., glucose, an alcohol, a ketone, lacate, β-hydroxybutyrate)over a time period which may range from seconds, minutes, hours, days,weeks, to months, or longer.

In certain embodiments, the analyte sensor includes a mass transportlimiting layer, e.g., an analyte flux modulating layer, to act as adiffusion-limiting barrier to reduce the rate of mass transport of theanalyte, for example, glucose, an alcohol, a ketone, lactate,β-hydroxybutyrate, when the sensor is in use. The mass transportlimiting layers limit the flux of an analyte to the electrode in anelectrochemical sensor so that the sensor is linearly responsive over alarge range of analyte concentrations. Mass transport limiting layersmay include polymers and may be biocompatible. A mass transport limitinglayer may provide many functions, e.g., biocompatibility and/orinterferent-eliminating functions, etc., or functions may be provided byvarious membrane layers.

In certain embodiments, a mass transport limiting layer is a membranecomposed of crosslinked polymers containing heterocyclic nitrogengroups, such as polymers of polyvinylpyridine and polyvinylimidazole.Embodiments also include membranes that are made of a polyurethane, orpolyether urethane, or chemically related material, or membranes thatare made of silicone, and the like.

The membrane may be formed by crosslinking in situ a polymer, modifiedwith a zwitterionic moiety, a non-pyridine copolymer component, andoptionally another moiety that is either hydrophilic or hydrophobic,and/or has other desirable properties, in an alcohol-buffer solution.The modified polymer may be made from a precursor polymer containingheterocyclic nitrogen groups. For example, a precursor polymer may bepolyvinylpyridine or polyvinylimidazole. Optionally, hydrophilic orhydrophobic modifiers may be used to “fine-tune” the permeability of theresulting membrane to an analyte of interest. Optional hydrophilicmodifiers, such as poly(ethylene glycol), hydroxyl or polyhydroxylmodifiers, may be used to enhance the biocompatibility of the polymer orthe resulting membrane.

Suitable mass transport limiting membranes in the subject analytesensors may include, but are not limited to those described in U.S. Pat.No. 6,932,894, the disclosure of which is herein incorporated byreference. In certain embodiments, the mass transport limiting membraneis a SMART membrane that is temperature independent. Suitabletemperature independent membranes may include, but are not limited tothose described in U.S. Patent Publication No. 2012/0296186 andcopending U.S. patent application Ser. No. 14/737,082, the disclosuresof which are herein incorporated by reference.

Analyte sensors according to certain embodiments may be configured tooperate at low oxygen concentration. By low oxygen concentration ismeant the concentration of oxygen is 1.5 mg/L or less, such as 1.0 mg/Lor less, such as 0.75 mg/L or less, such as 0.6 mg/L or less, such as0.3 mg/L or less, such as 0.25 mg/L or less, such as 0.15 mg/L or less,such as 0.1 mg/L or less and including 0.05 mg/L or less.

Aspects of the present disclosure also include methods for in vivomonitoring analyte levels over time with analyte sensors incorporatingan enzyme composition containing nicotinamide adenine dinucleotidephosphate (NAD(P)+) or derivative thereof, NAD(P)+-dependentdehydrogenase, NAD(P)H oxidoreductase and electron transfer agent.Generally, in vivo monitoring the concentration of analyte in a fluid ofthe body of a subject includes inserting at least partially under a skinsurface an in vivo analyte sensor as disclosed herein, contacting themonitored fluid (interstitial, blood, dermal, and the like) with theinserted sensor, and generating a sensor signal at the workingelectrode. The presence and/or concentration of analyte detected by theanalyte sensor may be displayed, stored, forwarded, and/or otherwiseprocessed. A variety of approaches may be employed to determine theconcentration of analyte (e.g., glucose, an alcohol, a ketone, lacate,β-hydroxybutyrate) with the subject sensors. In certain aspects, anelectrochemical analyte concentration monitoring approach is used. Forexample, monitoring the concentration of analyte using the sensor signalmay be performed by coulometric, amperometric, voltammetric,potentiometric, or any other convenient electrochemical detectiontechnique.

These methods may also be used in connection with a device that is usedto detect and/or measure another analyte, including glucose, oxygen,carbon dioxide, electrolytes, or other moieties of interest, forexample, or any combination thereof, found in a bodily fluid, includingsubcutaneous e.g., interstitial fluid, dermal fluid, blood or otherbodily fluid of interest or any combination thereof.

In certain embodiments, the method further includes attaching anelectronics unit to the skin of the patient, coupling conductivecontacts of the electronics unit to contacts of the sensor, collectingdata using the electronics unit regarding a level of analyte fromsignals generated by the sensor, and forwarding the collected data fromelectronics unit to a receiver unit, e.g., by RF. The receiver unit maybe a mobile telephone. The mobile telephone may include a applicationrelated to the monitored analyte. In certain embodiments, analyteinformation is forwarded by RFID protocol, Bluetooth, and the like.

The analyte sensor may be positionable in a user for automatic analytesensing, either continuously or periodically. Embodiments may includemonitoring the level of the analyte over a time period which may rangefrom seconds, minutes, hours, days, weeks, to months, or longer. Futureanalyte levels may be predicted based on information obtained, e.g., thecurrent lactate level at time zero as well as an analyte rate of change.

The sensor electronics unit may automatically forward data from thesensor/electronics unit to one or more receiver units. The sensor datamay be communicated automatically and periodically, such as at a certainfrequency as data is obtained or after a certain time period of sensordata stored in memory. For example, sensor electronics coupled to an invivo positioned sensor may collect the sensor data for a predeterminedperiod of time and transmit the collected data periodically (e.g., everyminute, five minutes, or other predetermined period) to a monitoringdevice that is positioned in range from the sensor electronics.

In other embodiments, the sensor electronics coupled to the in vivopositioned sensor may communicate with the receiving device nonautomatically manner and not set to any specific schedule. For example,the sensor data may be communicated from the sensor electronics to thereceiving device using RFID technology, and communicated whenever thesensor electronics are brought into communication range of the analytemonitoring device. For example, the in vivo positioned sensor maycollect sensor data in memory until the monitoring device (e.g.,receiver unit) is brought into communication range of the sensorelectronics unit—e.g., by the patient or user. When the in vivopositioned sensor is detected by the monitoring device, the deviceestablishes communication with the analyte sensor electronics anduploads the sensor data that has been collected since the last transferof sensor data, for instance. In this way, the patient does not have tomaintain close proximity to the receiving device at all times, andinstead, can upload sensor data when desired by bringing the receivingdevice into range of the analyte sensor. In yet other embodiments, acombination of automatic and non-automatic transfers of sensor data maybe implemented in certain embodiments. For example, transfers of sensordata may be initiated when brought into communication range, and thencontinued on an automatic basis if continued to remain in communicationrange.

EXPERIMENTAL

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the embodiments of the invention, and are not intended tolimit the scope of what the inventors regard as their invention nor arethey intended to represent that the experiments below are all or theonly experiments performed. Efforts have been made to ensure accuracywith respect to numbers used (e.g., amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

Example 1

Experiments were performed to demonstrate the performance of analytesensors having a working electrode that contains nicotinamide adeninedinucleotide phosphate (NAD(P)⁺) or derivative thereof,NAD(P)⁺-dependent dehydrogenase, NAD(P)H oxidoreductase and electrontransfer agent. The sensors were prepared by depositing onto the surfaceof an electrode an enzyme composition containing nicotinamide adeninedinucleotide phosphate, D-3-hydroxybutyrate dehydrogenase, diaphoraseand a polymer bound osmium-transition metal catalyst and a difunctionalcrosslinker, as shown by the scheme (also referred to as a polymericredox mediator in further examples):

The sensors were tested in phosphate buffer containing varyingconcentrations of D-3-hydroxybutyrate. Table 1 summarizes beakercalibration and linearity of data signal from the prepared sensors.

TABLE 1 D-3-Hydroxybutyrate Sensor Slope 0.0163 R² 0.9982

FIG. 1 shows the signal output over the course of 2.3 hours at varyingconcentrations of D-3-hydroxybutyrate (80 μM, 160 μM and 240 μM). FIG. 2depicts the linearity of the sensor signal as a function ofD-3-hydroxybutyrate concentration. As shown in FIGS. 1 and 2, the sensorgives a linear and persistent response to D-3-hydroxybutyrate.

Example 2

Experiments were performed to demonstrate the performance of analytesensors having a working electrode that contains free NAD. The sensorwas prepared by depositing onto the surface of an electrode an enzymecomposition containing the free NAD. Sensing layer formulation isdescribed in Table 2. The sensing layer solutions was deposit on carbonelectrodes, and cured at 25 C/60 H overnight, prior to addition ofmembrane. The membrane formulation is provided in Table 3. The sensorwas dipped from above solution 3×5 mm/sec, and the sensor was cured at25 C/60 H overnight, 56 C for two days.

TABLE 2 Formulation Table Sensing Layer Solution BD161212 Mixing Final10 mM Hepes Vendor Cat mg/mL uL mg/mL D-3-Hydroxybutyrate Toyobo HBD- 4020 8.89 Dehydrogenase (HBD) 301 Diaphorase Toyobo DAD- 40 20 8.89 311NAD Sigma N0632 20 10 2.22 Polymeric redox 40 20 8.89 mediator Peg400 4020 8.89 Total 90

TABLE 3 Membrane Coating Membrane Solution Solution A Vendor Cat MWMixing Poly(4-vinylpyridine) Sigma 472352 160K 120 mg Ethanol/Hepe (10mM pH 8.0)  1 mL Solution B Vendor Cat Mixing Poly(ethylene glycol)diglycidyl ether Polysciences 8210 100 mg (PEGDGE 400) Ethanol/Hepe (10mM pH 8.0)  1 mL Final membrane solution Mixing (mL) Solution A 4Solution B 0.4

FIG. 3 shows the signal output over the course of 3.6 hours at varyingconcentrations of D-3-hydroxybutyrate (ketone). FIG. 4 depicts thelinearity of the sensor signal as a function of D-3-hydroxybutyrateconcentration. FIG. 5 shows sensor calibration at 10 mM. As shown inFIGS. 3-5, the sensor gives a linear and persistent response toD-3-hydroxybutyrate (ketone). Table 4 also shows the free NAD KetoneSensor Beaker Calibration and Stability Summary.

TABLE 4 Slope 2.55 R{circumflex over ( )}2 0.9996 Decay in 45 hours −8%

Example 3

Experiments were also performed to demonstrate the performance of ketonesensors having a working electrode that contains free NAD versusimmobilized NAD. The sensors were prepared by depositing onto thesurface of an electrode an enzyme composition containing the free NAD(A) or the immobilized NAD (B). Sensing layer formulation is describedin Table 5. The sensing layer solutions was deposit on carbonelectrodes, and cured at 25 C/60 H overnight, prior to addition ofmembrane. The membrane formulation is provided in Table 6. The sensorwas dipped (Table 7) from above solution 3×5 mm/sec, and the sensor wascured at 25 C/60 H overnight, 56 C for two days. FIG.6 shows that boththe free and immobilized NAD versions of the ketone sensor show similarstabilities and signals.

TABLE 5 Sensing Layer Solution Mixing Final BD161116 uL mg/mL 10 mMHepes Vendor Cat mg/mL A B A B D-3- Toyobo HBD-301 40 20 20 8.89 8.89Hydroxybutyrate Dehydrogenase (HBD) Diaphorase Toyobo DAD-311 40 20 208.89 8.89 NAD Sigma N0632 20 10 2.22 0.00 NAD-NH2 Biotium 40 10 0.004.44 Glutaraldehyde 10 0 5 0.00 0.56 Polymeric redox 40 20 20 8.89 8.89mediator Peg400 40 20 20 8.89 8.89 ToTal 90 95

TABLE 6 Membrane Coating Membrane Solution Solution A Vendor MW MixingSmart Membrane ADC 160K 120 mg Ethanol/Hepe (10 mM pH 8.0)  1 mLSolution B Vendor Cat Mixing Poly(ethylene glycol) diglycidyl etherPolysciences 8210 100 mg (PEGDGE 400) Ethanol/Hepe (10 mM pH 8.0)  1 mLFinal membrane solution Mixing (mL) Solution A 4 Solution B 0.35

TABLE 7 Membrane Dipping Condition (Free NAD sensor was coated thickermembrane than Immobilized NAD sensor) SL Solution Dipping A (Free NAD) 4× 5 B (Immobilized NAD) 3 × 5

That which is claimed is:
 1. An enzyme composition comprising:nicotinamide adenine dinucleotide phosphate (NAD(P)⁺) or derivativethereof; an NAD(P)⁺-dependent dehydrogenase; an NAD(P)H oxidoreductase;and an electron transfer agent comprising a transition metal complex,wherein the nicotinamide adenine dinucleotide phosphate (NAD(P)⁺) orderivative thereof, the NAD(P)⁺-dependent dehydrogenase, the NAD(P)Hoxidoreductase, and the electron transfer agent comprising thetransition metal complex are distributed within a single polymercomposition.
 2. The enzyme composition according to claim 1, wherein thecomposition comprises nicotinamide adenine dinucleotide phosphate(NAD(P)⁺).
 3. The enzyme composition according to claim 1, wherein thecomposition comprises a derivative of nicotinamide adenine dinucleotidephosphate.
 4. The enzyme composition according to claim 3, wherein thederivative of nicotinamide adenine dinucleotide phosphate is a compoundof Formula I:

wherein X is alkyl, substituted alkyl, aryl, substituted aryl, acyl, andaminoacyl.
 5. The enzyme composition according to claim 4, wherein thederivative of nicotinamide adenine dinucleotide phosphate is:


6. The enzyme composition according to claim 1, wherein theNAD(P)⁺-dependent dehydrogenase is selected from the group consisting ofglucose dehydrogenase, alcohol dehydrogenase, D-3-hydroxybutyratedehydrogenase.
 7. The enzyme composition according to claim 1, whereinthe NAD(P)H oxidoreductase is diaphorase.
 8. The enzyme compositionaccording to claim 1, wherein electron transfer agent comprises anosmium-containing complex.
 9. The enzyme composition according to claim8, wherein the osmium-containing complex comprises three heterocyclicnitrogen-containing bidentate ligands.
 10. The enzyme compositionaccording to claim 9, wherein the osmium-containing complex comprisestwo biimidazole and one imidazole-pyridine bidentate ligands.
 11. Theenzyme composition according to claim 1, wherein the polymer compositioncomprises a heterocycle-containing polymer and a crosslinker.
 12. Theenzyme composition according to claim 11, wherein theheterocycle-containing polymer comprises polyvinylpyridine.
 13. Theenzyme composition according to claim 11, wherein at least one of thenicotinamide adenine dinucleotide phosphate (NAD(P)⁺) or derivativethereof; the NAD(P)⁺-dependent dehydrogenase; the NAD(P)Hoxidoreductase; and the electron transfer agent comprising thetransition metal complex are immobilized by the heterocycle-containingpolymer.
 14. The enzyme composition according to claim 13, wherein atleast one of the nicotinamide adenine dinucleotide phosphate (NAD(P)⁺)or derivative thereof; NAD(P)⁺-dependent dehydrogenase; NAD(P)Hoxidoreductase; and redox mediator comprising the transition metalcomplex are covalently bonded to the heterocycle-containing polymer. 15.The enzyme composition according to claim 14, wherein the electrontransfer agent comprising the transition metal complex is covalentlybonded to the heterocycle-containing polymer.
 16. The enzyme compositionaccording to claim 1, comprising: nicotinamide adenine dinucleotidephosphate (NAD(P)⁺) or derivative thereof; glucose dehydrogenase;diaphorase; and an osmium-containing electron transfer agent.
 17. Theenzyme composition according to claim 1, comprising: nicotinamideadenine dinucleotide phosphate (NAD(P)⁺) or derivative thereof; alcoholdehydrogenase; diaphorase; and an osmium-containing electron transferagent.
 18. The enzyme composition according to claim 1, comprising:nicotinamide adenine dinucleotide phosphate (NAD(P)³⁰ ) or derivativethereof; D-3-hydroxybutyrate dehydrogenase; diaphorase; and anosmium-containing electron transfer agent.
 19. An enzyme compositioncomprising: nicotinamide adenine dinucleotide phosphate (NAD(P)⁺) orderivative thereof; an NAD(P)⁺-dependent dehydrogenase; an NAD(P)Hoxidoreductase; and an electron transfer agent comprising a transitionmetal complex, wherein the nicotinamide adenine dinucleotide phosphate(NAD(P)⁺) or derivative thereof, the NAD(P)⁺-dependent dehydrogenase,the NAD(P)H oxidoreductase, and the electron transfer agent comprisingthe transition metal complex are uniformly distributed within a singlepolymer composition.